Hi I have a question in setting up a control for background staining when doing IHC.
I have a polyclonal antibody to stain tissue using IHC. I was going to use isotype control. However, it seems like isotype controls are used when using monoclonal antibody in IHC?
I do not understand why? In my mind, polyclonal antibodies would have strong signal due to the ability of recognizing multiple epitopes of an antigen compared to monoclonal antibodies. Then should polyclonal antibody staining also have an isotype control? If not, why people do not do that and what is the alternative for background staining when doing IHC with polyclonal antibodys?
Thanks!
Good day, dear Bumjun Kim .
Nobody doing isotype control due to the fact that polyclonal antibodies for IHC mostly produced by hybridomas in the peritoneal cavities of lab animals, so it could have a huge number of isotypes. For control of policlonal antibodies mostly used positibe and negative control. Positive control : you take different tissue that expressed 100% this antibody, for example angiosarcoma tissue for CD34. Negative control: you do IHC but dont add primary antibody on one slide.
Also in my lab we do antigen unmask control. We stain one slide of lymph node with reactive hyperplasia for Ki-67. If it is possitive antigen unmask was done correct.
Best regards,
Thank you so much Dr Zinovikin.
I will include a positive and negative controls for sure.
Thanks for your advice again.
Best,
Bumjun
Well, I've been performing isotype controles for pAbs as well during my 35yrs with IHC, but it is discussabel for several reasons. As Dmitry says, a positive controle slide should be included to validate the antibody. A negative (PBS) controle should not stain. Isotype-matched controles:
1) your antibody has probably been purified in a different way than your isotype controles, if purified at all. So they will probably not be similar.
2) my experience tells me that the best controle is pre-immune serum from the same animal. I always get cleaner results then commercial available isotype controles. One reason can be that the pooled serum comes from animals that might have had an infection earlier. They are stated as "healthy" when they are bleeded, but.... So do not be surprised when the commercial isotype controle leads to more unspecific staining. Solution: test others or other batches.That is true for antibodies as well, but be aware that the same polyclonal can be sold through different companies (check the pictures; WB and IHC will never give the same picture when you redo it, even with the same antibody).
3) Use another polyclonal antibody of the same isotype class and purified in a similar way (might be different) and similar concentration if known. You might have internal controles already (another pAb towards another molecule). If not, use another antibody you know well, e.g. Ki-67 that only binds inside nucleï, and convince yourself that the are different and do no stain similar unspecific structures.
Good luck!
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